Bacillus anthracis protective antigen sequences

ABSTRACT

The invention relates to improved methods of producing and recovering  B. anthracis  protective antigen (PA), especially modified PA which is protease resistant, and to methods of using of these PAs or nucleic acids encoding these PAs for eliciting an immunogenic response in humans, including responses which provide protection against, or reduce the severity of,  B. anthracis  bacterial infections and which are useful to prevent and/or treat illnesses caused by  B. anthracis , such as inhalation anthrax, cutaneous anthrax and gastrointestinal anthrax.

FIELD OF THE INVENTION

This invention relates to improved methods of preparing Bacillusanthracis protective antigen (PA) for use in vaccines.

BACKGROUND

Anthrax, a potentially fatal disease, is caused by Bacillus anthracis.The virulence of this pathogen is mediated by a capsule of apoly-D-γ-glutamic acid and an exotoxin composed of three proteins (14,16, 17). The three protein components are the protective antigen (PA, 82KDa), lethal factor (LF, 90.2 KDa) and edema factor (EF, 88.8 KDa) Theseproteins, non-toxic by themselves, form lethal toxins when combined withan activated PA (16). The genes coding for these three proteincomponents and the capsule are found in the endogenous plasmids pXO1 andpXO2, respectively (29).

The capsule of Bacillus anthracis, composed of poly-D-glutamic acid,serves as one of the principal virulence factors during anthraxinfection. By virtue of its negative charge, the capsule is purported toinhibit host defense through inhibition of phagocytosis of thevegetative cells by macrophages. In conjunction with lethal factor (LF)and edema factor (EF), whose target cells include macrophages andneutrophils, respectively, the capsule allows virulent anthrax bacillito grow virtually unimpeded in the infected host. Spores germinating inthe presence of serum and elevated CO₂ release capsule through openingson the spore surface in the form of blebs which may coalesce beforesloughing of the exosporium and outgrowth of the fully encapsulatedvegetative cell. It has not been established that spore encapsulationplays a role in the early events of anthrax infection. The capsuleappears exterior to the S-layer of the vegetative cell and does notrequire the S-layer for its attachment to the cell surface.

There is only indirect evidence, albeit extensive, identifying thecomponents of vaccine-induced immunity to anthrax and there is evidencethat anti-PA neutralizing antibody titers can be a reliable surrogatemarker for protective immunity (23). The protective antigen (PA), seemsto be an essential component of all vaccines for anthrax (7, 18, 30):both mono and polyclonal antibodies to PA neutralize the anthrax toxinand confer immunity to B. anthracis in animal models. The US licensedvaccine for anthrax “Anthrax Vaccine Adsorbed” (AVA) is produced fromthe formalin-treated culture supernatant of B. anthracis Sterne strain,V770-NP1-R (pXO1⁺, pXO2⁻), adsorbed onto aluminum hydroxide (22).Although AVA has been shown to be effective against cutaneous infectionin animals and humans and against inhalation anthrax by rhesus monkeys(12), it has several limitations: 1) AVA elicits relatively high degreeof local and systemic adverse reactions probably mediated by variableamounts of undefined bacterial products, making standardizationdifficult; 2) the immunization schedule requires administration of sixdoses within an eighteen-month period, followed by annual boosters forthose at risk; and 3) there is no defined vaccine-induced protectivelevel of serum PA to evaluate new lots of vaccines.

Development of a well characterized, standardized, effective and safevaccine that would require fewer doses to confer immunity to bothinhalational and cutaneous anthrax is needed (9, 30). It has beensuggested that a vaccine composed of modified purified recombinant PAwould be effective, safer, allow precise standardization, and probablywould require fewer injections (27). Such a PA can be designed to bebiologically inactive, more stable, and still maintained highimmunogenicity.

In the examples herein, we describe the development of a production andpurification process for recombinant PA from the non-sporogenicavirulent B. anthracis BH445 (pXO1⁻, pXO2⁻) strain. Following an 18-hourfermentation and three purification steps, large quantities ofprotective antigen suitable for vaccine production were obtained. Thepurified PA was tested in mice and was able to elicit neutralizingantibodies.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to improved methods of preparing Bacillusanthracis protective antigen (PA).

The invention also relates to PA and/or compositions thereof, which areuseful for inducing or eliciting an immunogenic response in mammals,including responses which provide protection against, or reduce theseverity of, infections caused by B. anthracis. In particular, theinvention relates to methods of using PA, and/or compositions thereof,to induce or elicit serum antibodies which have neutralizing activityagainst B. anthracis toxin. PA and/or compositions thereof are useful asvaccines to induce serum antibodies which are useful to prevent, treator reduce the severity of infections caused by B. anthracis, such asinhalation anthrax, cutaneous anthrax and/or gastrointestinal anthrax.

The invention also relates to nucleic acids encoding PA of B. anthracis,and compositions thereof, which produce PA in sufficient amounts to beuseful as pharmaceutical compositions or vaccines to induce serumantibodies for preventing and/or treating illnesses caused by B.anthracis. The invention also relates to suitable expression systems,viral particles, vectors, vector systems, and transformed host cellscontaining those nucleic acids.

The invention also relates to antibodies which immunoreact with the PAof B. anthracis, and/or compositions thereof. Such antibodies may beisolated, or may be provided in the form of serum containing theseantibodies.

The invention also relates to pharmaceutical compositions and/orvaccines comprising at least one of the PAs, nucleic acids, viralparticles, vectors, vector systems, transformed host cells or antibodiesof the invention.

The invention also relates to methods for the prevention or treatment ofB. anthracis infection in a mammal, by administration of pharmaceuticalor vaccine compositions of the invention.

The invention also provides kits comprising one or more of the agents ofthe invention which are useful for vaccinating mammals for the treatmentor prevention of B. anthracis infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Production and proteolytic activity of PA-SNKE-ΔFF-E308D andPA-N657A. (a) PA production (mg/g cells)  SNKE, ▪ N657A; proteolyticactivity ◯ SNKE, □ N657A; (b) SDS-PAGE analysis of partially purifiedPA-N657A and PA-SNKE-ΔFF-E308D.

FIG. 2. Effect of EDTA and PMSF on proteolytic activity. Supernatantsfrom two different cultures taken after 24 hours of growth were analyzedwithout inhibitors (control), with 1 μg/μL PMSF, and with 15 mM EDTA.Fluorescence is proportional to proteolytic activity.

FIG. 3. Fermentation process for the production of PA-SNKE-ΔFF-E308Dfrom B. anthracis BH445. Acid and base values are cumulative.

FIG. 4. SDS-PAGE analysis of culture supernatants obtained throughoutthe fermentation. Samples were taken at 13, 14, 16, 18, 22, and 34 hoursof growth. Arrow indicates the location of PA(83 KDa) in the gel.

FIG. 5. PA production and proteolytic activity of B. anthracis BH445[pSY5:SNKE-ΔFF-E308D] in fed-batch cultures supplied with tryptone/yeastextract or glucose.  Specific PA production in tryptone/yeast extract(mg/g cells); ▪ Volumetric PA production in tryptone/yeast extract(mg/liter); ▴ Proteolytic activity in tryptone/yeast extract; ◯ SpecificPA production in glucose (mg/g cells); □ Volumetric PA production inglucose (mg/liter); A Proteolytic activity in glucose.

FIG. 6. SDS-PAGE analysis of purified PA fractions. (a) PA purified bypacked bed chromatography; (b) PA after hydrophobic interactionchromatography and gel filtration; (c) PA fraction shown in Lane (b)after 3 months; (d) PA after expanded bed hydrophobic interactionchromatography, anion exchange, and gel filtration. MW indicatesmolecular weight markers. Arrows indicate the location of PA(83 KDa) inthe gel.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only,and are not restrictive of the invention, as claimed. The accompanyingdrawings, which are incorporated in and constitute a part of thespecification, illustrate an embodiment of the invention and, togetherwith the description, serve to explain the principles of the invention.

The invention relates to methods of producing and recovering PA from acell or organism, particularly a recombinant cell or microorganism.Exemplified herein is the production and purification of modified PAfrom a non-sporgenic strain of Bacillus anthracis. As discussed furtherherein, greater quantities of PA are obtainable from these cells ormicroorganisms than were obtainable by previously described methods.

The invention also relates to PA, and/or compositions thereof, which areuseful for eliciting an immunogenic response in mammals, in particularhumans, including responses which provide protection against, or reducethe severity of, infections caused by B. anthracis. The invention alsorelates to methods of using such PA, and/or compositions thereof, toinduce serum antibodies against PA. PA, and/or compositions thereof, areuseful as vaccines to induce serum antibodies which are useful toprevent, treat or reduce the severity of infections caused by B.anthracis, such as inhalation anthrax and/or cutaneous anthrax. The PAsof this invention are expected to induce a strong protective IgGantibody response in mammals, including humans.

The invention also relates to nucleic acids encoding PA of thisinvention. Nucleic acids encoding PA, and compositions thereof, are alsouseful as pharmaceutical compositions or vaccines to induce serumantibodies which are useful to prevent and/or treat illnesses caused byB. anthracis.

The invention also relates to antibodies which immunoreact with the PAof B. anthracis that are induced by PAs of the invention, and/orcompositions thereof. Such antibodies may be isolated, or may beprovided in the form of serum containing these antibodies.

The invention also relates to a method for the prevention or treatmentof B. anthracis infection in a mammal, by administration of compositionscontaining one or more of a PA of the invention, nucleic acids encodinga PA if the invention, antibodies and/or serum containing antibodies ofthe invention.

The invention also provides kits for vaccinating mammals for thetreatment or prevention of B. anthracis infection in a mammal comprisingone or more of the agents of the invention.

The present invention also encompasses methods of using mixtures of oneor more of the PA, nucleic acids, and/or antibodies of the invention,either in a single composition or in multiple compositions containingother immunogens, to form multivalent vaccine for broad coverage againsteither B. anthracis itself or a combination of B. anthracis and one ormore other pathogens, which may also be administered concurrently withother vaccines, such as the DTP vaccine.

Pharmaceutical compositions of this invention are capable, uponinjection into a human, of inducing serum antibodies against B.anthracis. The induced anti-PA antibodies have anthrax toxinneutralizing activity which are preferably at least comparable to thoseinduced by the currently licensed anthrax vaccine.

The vaccines of this invention are intended for active immunization forprevention of B. anthracis infection, and for preparation of immuneantibodies. The vaccines of this invention are designed to conferspecific immunity against infection with B. anthracis, and to induceantibodies specific to B. anthracis PA. The B. anthracis vaccine iscomposed of non-toxic bacterial components, suitable for infants,children of all ages, and adults.

The methods of using the agents of this invention, and/or compositionsthereof will be useful in increasing resistance to, preventing,ameliorating, and/or treating B. anthracis infection in humans.

This invention also provides compositions, including but not limited to,mammalian serum, plasma, and immunoglobulin fractions, which containantibodies which are immunoreactive with B. anthracis PA. Theseantibodies and antibody compositions may be useful to prevent, treat,and/or ameliorate infection and disease caused by the microorganism. Theinvention also provides such antibodies in isolated form.

High titer anti-PA sera, or antibodies isolated therefrom, may be usedfor therapeutic treatment for patients with B. anthracis infection.Antibodies elicited by the agents of this invention may be used for thetreatment of established B. anthracis infections, and may also be usefulin providing passive protection to an individual exposed to B.anthracis.

The present invention also provides kits comprising vaccines for theprevention and/or treatment of B. anthracis, containing the one or moreof the PAs, nucleic acids, viral particles, vectors, vector systems, ortransformed host cells or antibodies of the invention and/orcompositions thereof. The PAs, nucleic acids viral particles vectors,host cells and/or antibodies of the present invention may be isolatedand purified by methods known in the art. Preferably, the PA of theinvention is purified by one of the methods exemplified herein.

The vaccines of the invention are intended to be included in theimmunization schedule of individuals at risk for B. anthracis infection.They are also planned to be used for intervention in the event of theuse of B. anthracis in bioterrorism or biowarfare. For example, it isanticipated that the vaccines of the invention may be provided to theentire U.S. population. Additionally, they may be used as component(s)of a multivalent vaccine for B. anthracis and/or other pathogens.

DEFINITIONS

As used herein, unless otherwise specifically noted, “PA” refers to allforms of PA which are useful in the compositions and/or methods of theinvention, including unmodified native or recombinant B. anthracisprotective antigen (PA), or a modified form (variant) or fragmentthereof, for use in vaccines. Variants and fragments of PA must be ableto produce an immune response in a mammal to whom they are administered.The immune response is suitably protective against infection by Bacillusanthracis although the protective effect may be seen only after repeatedapplications, as would be determinable by methods known in the art.Modified PA variants comprise peptides and proteins which resemble PA intheir ability to induce or elicit antibodies which bind to native PA,but have different amino acid sequence. For example, variants may be 60%homologous to PA protein, suitably 80% homologous and more particularlyat least 90% homologous. Fragments are suitably peptides which containat least one antigenic determinant of PA.

A modified (variant) PA of the invention includes any substituted analogor chemical derivative of PA, so long as the modified (variant) PA iscapable of inducing or eliciting the production of antibodies capable ofbinding native (or naturally-occurring) PA. Preferably, the antibodiesare neutralizing antibodies. PA can be subject to various changes thatprovide for certain advantages in its use. For example, PA with changeswhich increase in vitro and/or in vivo stability of PA, while stillretaining the desired immunogenic activity, are preferred. In themodified PA used in the examples herein, two regions were altered, i.e.,the furin cleavage site region (RKKR167 to SNKE167), and thechymotrypsin and thermolysin cleavage site region (two Phe at positions313-314 were deleted and Glu acid at position 308 was substituted withAsp), resulting in a more stable PA. As used herein, the terms“immunoreact” and “immunoreactivity” refer to specific binding betweenan antigen or antigenic determinant-containing molecule and a moleculehaving an antibody combining site, such as a whole antibody molecule ora portion thereof.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules.Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and portions of animmunoglobulin molecule, including those portions known in the art asFab, Fab′, F(ab′)₂ and F(v), as well as chimeric antibody molecules.

As used herein, the term “transduction” generally refers to the transferof genetic material into the host via infection, e.g., in this case bythe lentiviral vector. The term “transfection” generally refers to thetransfer of isolated genetic material into cells via the use of specifictransfection agents (e.g., calcium phosphate, DEAE Dextran, lipidformulations, gold particles, and other microparticles) that cross thecytoplasmic membrane and deliver some of the genetic material into thecell nucleus.

Monomers, Polymers and Polymeric Carriers

The present invention encompasses monomers of PA, as well as homogeneousor heterogeneous polymers of PA (e.g., concatenated, cross-linked and/orfused identical polypeptide units or concatenated, cross-linked and/orfused diverse peptide units), and mixtures of the polypeptides,polymers, and/or conjugates thereof. The present invention alsoencompasses PA bound to a non-toxic, preferably non-host, proteincarrier to form a conjugate.

Linkers useful in the invention may, for example, be simply peptidebonds, or may comprise amino acids, including amino acids capable offorming disulfide bonds, but may also comprise other molecules such as,for example, polysaccharides or fragments thereof.

The linkers for use with this invention may be chosen so as tocontribute their own immunogenic effect which may be either the same, ordifferent, than that elicited by the consensus sequences of theinvention. For example, such linkers may be bacterial antigens whichalso elicit the production of antibodies to infectious bacteria. In suchinstances, for example, the linker may be a protein or protein fragmentof an infectious bacteria.

Carriers are chosen to increase the immunogenicity of the PA and/or toraise antibodies against the carrier which are medically beneficial.Carriers that fulfill these criteria are well known in the art. Apolymeric carrier can be a natural or a synthetic material containingone or more functional groups, for example primary and/or secondaryamino groups, azido groups, or carboxyl groups. Carriers can be watersoluble or insoluble.

Methods for Attaching PA to a Protein Carrier.

PA of the invention may be covalently attached to other proteins, withor without a linker, by methods known in the art, such as via their sidechains or via peptide bonds in the primary chain. Cysteine molecules mayprovide a convenient attachment point through which to chemicallyconjugate other proteins or non-protein moieties to PA.

Dosage for Vaccination

The pharmaceutical compositions of this invention contain apharmaceutically and/or therapeutically effective amount of at least onePA, nucleic acid, vector, viral particle, host cell immunogen orantibody of the invention. The effective amount of immunogen per unitdose is an amount sufficient to induce an immune response which issufficient to prevent, treat or protect against the adverse effects ofinfection with B. anthracis. The effective amount of immunogen per unitdose depends, among other things, on the species of mammal inoculated,the body weight of the mammal and the chosen inoculation regimen, as iswell known in the art.

In such circumstances, inocula for a human or similarly sized mammaltypically contain PA concentrations of 0.5 μg to 1 mg per mammal perinoculation dose. Initial tests of the PA vaccine in humans will useapproximately 10 μg or 20 μg per dose. Preferably, the route ofinoculation of the peptide will be subcutaneous or intramuscular. Thedose is administered at least once.

To monitor the antibody response of individuals administered thecompositions of the invention, antibody levels may be determined. Inmost instances it will be sufficient to assess the antibody titer inserum or plasma obtained from such an individual. Decisions as towhether to administer booster inoculations or to change the amount ofthe composition administered to the individual may be at least partiallybased on the level.

The level may be based on either an immunobinding assay which measuresthe concentration of antibodies in the serum which bind to a specificantigen, i.e. PA. The ability to neutralize in vitro and in vivobiological effects of the B. anthracis toxins may also be assessed todetermine the effectiveness of the treatment.

The term “unit dose” as it pertains to the inocula refers to physicallydiscrete units suitable as unitary dosages for mammals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired immunogenic effect in association with the requireddiluent.

Inocula are typically prepared in physiologically and/orpharmaceutically tolerable (acceptable) carrier, and are preferablyprepared as solutions in physiologically and/or pharmaceuticallyacceptable diluents such as water, saline, phosphate-buffered saline, orthe like, to form an aqueous pharmaceutical composition. Adjuvants, suchas aluminum hydroxide, may also be included in the compositions.

Depending on the intended mode of administration, the compounds of thepresent invention can be in various pharmaceutical compositions. Thecompositions will include, as noted above, an effective amount of theselected immunogen and/or antibody of the invention in combination witha pharmaceutically acceptable carrier and, in addition, may includeother medicinal agents, pharmaceutical agents, carriers, adjuvants,diluents, etc. By “pharmaceutically acceptable” is meant a material thatis not biologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the immunogen and/or antibodyor other composition without causing any undesirable biological effectsor interacting in a deleterious manner with any of the other componentsof the pharmaceutical composition in which it is contained.

The route of inoculation may be intramuscular, subcutaneous or the like,which results in eliciting antibodies protective against B. anthracis.In order to increase the antibody level, a second or booster dose may beadministered approximately 4 to 6 weeks after the initial injection.Subsequent doses may be administered as indicated herein, or as desiredby the practitioner.

Parenteral administration, if used, is generally characterized byinjection. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. A morerecently revised approach for parenteral administration involves use ofa slow release or sustained release system, such that a constant levelof dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which isincorporated by reference herein.

Antibodies

An antibody of the present invention in one embodiment is characterizedas comprising antibody molecules that immunoreact with B. anthracis PA.

An antibody of the present invention is typically produced by immunizinga mammal with an immunogen or vaccine containing an B. anthracis PA toinduce, in the mammal, antibody molecules having immunospecificity forthe immunizing PA. Antibody molecules having immunospecificity for theprotein carrier will also be produced. The antibody molecules may becollected from the mammal and, optionally, isolated and purified bymethods known in the art.

Human or humanized monoclonal antibodies are preferred, including thosemade by phage display technology, by hybridomas, or by mice with humanimmune systems. The antibody molecules of the present invention may bepolyclonal or monoclonal. Monoclonal antibodies may be produced bymethods known in the art. Portions of immunoglobulin molecules, such asFabs, may also be produced by methods known in the art.

The antibody of the present invention may be contained in blood plasma,serum, hybridoma supernatants and the like. Alternatively, theantibodies of the present invention are isolated to the extent desiredby well known techniques such as, for example, ion exchangechromatography, sizing chromatography, or affinity chromatography. Theantibodies may be purified so as to obtain specific classes orsubclasses of antibody such as IgM, IgG, IgA, IgG₁, IgG₂, IgG₃, IgG₄ andthe like. Antibodies of the IgG class are preferred for purposes ofpassive protection. The antibodies of the present invention have anumber of diagnostic and therapeutic uses. The antibodies can be used asan in vitro diagnostic agents to test for the presence of B. anthracisin biological samples or in meat and meat products, in standardimmunoassay protocols. Such assays include, but are not limited to,agglutination assays, radioimmunoassays, enzyme-linked immunosorbentassays, fluorescence assays, Western blots and the like. In one suchassay, for example, the biological sample is contacted first withantibodies of the present invention which bind to B. anthracis PA, andthen with a labeled second antibody to detect the presence of B.anthracis to which the first antibodies have bound.

Such assays may be, for example, of direct format (where the labeledfirst antibody is reactive with the antigen), an indirect format (wherea labeled second antibody is reactive with the first antibody), acompetitive format (such as the addition of a labeled antigen), or asandwich format (where both labeled and unlabelled antibody areutilized), as well as other formats described in the art. The antibodiesof the present invention are also useful in prevention and treatment ofinfections and diseases caused by B. anthracis.

In providing the antibodies of the present invention to a recipientmammal, preferably a human, the dosage of administered antibodies willvary depending upon such factors as the mammal's age, weight, height,sex, general medical condition, previous medical history and the like.

In general, it is desirable to provide the recipient with a dosage ofantibodies which is in the range of from about 1 mg/kg to about 10 mg/kgbody weight of the mammal, although a lower or higher dose may beadministered. The antibodies of the present invention are intended to beprovided to the recipient subject in an amount sufficient to prevent, orlessen or attenuate the severity, extent or duration of the infection byB. anthracis. When proteins of other organisms are used as carriers,antibodies which immunoreact with those proteins are intended to beprovided to the recipient subject in an amount sufficient to prevent,lessen or attenuate the severity, extent or duration of an infection bythe organisms producing those proteins.

The administration of the agents of the invention may be for either“prophylactic” or “therapeutic” purpose. When provided prophylactically,the agents are provided in advance of any symptom. The prophylacticadministration of the agent serves to prevent or ameliorate anysubsequent infection. When provided therapeutically, the agent isprovided at (or shortly after) the onset of a symptom of infection. Theagent of the present invention may, thus, be provided prior to theanticipated exposure to B. anthracis, so as to attenuate the anticipatedseverity, duration or extent of an infection and disease symptoms, afterexposure or suspected exposure to these bacteria, or after the actualinitiation of an infection.

For all therapeutic, prophylactic and diagnostic uses, one or more ofthe PAs or other agents of this invention, as well as antibodies andother necessary reagents and appropriate devices and accessories, may beprovided in kit form so as to be readily available and easily used.

Nucleic Acids, Vectors and Hosts

The invention also relates to isolated and purified nucleic acidmolecules which code for the PAs of the invention. The encoded PAs maybe monomers, polymers or linked to other peptide sequences (e.g., theymay be fusion proteins).

Nucleic acids encoding the PAs of the invention can be introduced into avector such as a plasmid, cosmid, phage, virus, viral particle ormini-chromosome and inserted into a host cell or organism by methodswell known in the art. The vectors which can be utilized to clone and/orexpress these nucleic acids are the vectors which are capable ofreplicating and/or expressing the nucleic acids in the host cell inwhich the nucleic acids are desired to be replicated and/or expressed.See, e.g., F. Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley-Interscience (1992) and Sambrooket al. (1989) for examples of appropriate vectors for various types ofhost cells. Vectors and compositions for enabling production of thepeptides in vivo, i.e., in the individual to be treated or immunized,are also within the scope of this invention. Strong promoters compatiblewith the host into which the gene is inserted may be used. Thesepromoters may be inducible. The host cells containing these nucleicacids can be used to express large amounts of the protein useful inpharmaceuticals, diagnostic reagents, vaccines and therapeutics. Vectorsinclude retroviral vectors and also include direct injection of DNA intomuscle cells or other receptive cells, resulting in the efficientexpression of the peptide, using the technology described, for example,in Wolff et al., Science 247:1465-1468 (1990), Wolff et al., HumanMolecular Genetics 1(6):363-369 (1992) and Ulmer et al., Science259:1745-1749 (1993). See also, for example, WO 96/36366 and WO98/34640.

In general, vectors containing nucleic acids encoding PA can be utilizedin any cell, either eukaryotic or prokaryotic, including mammalian cells(e.g., human (e.g., HeLa), monkey (e.g., COS), rabbit (e.g., rabbitreticulocytes), rat, hamster (e.g., CHO and baby hamster kidney cells)or mouse cells (e.g., L cells), plant cells, yeast cells, insect cellsor bacterial cells (e.g., E. coli). However, bacterial vectors and hostcells are preferred in the present invention.

There are numerous E. coli expression vectors known to one of ordinaryskill in the art useful for the expression of PA. Other microbial hostssuitable for use include bacilli, such as B. subtilus, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (Trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences for example, for initiating and completingtranscription and translation. If necessary an amino terminal methioninecan be provided by insertion of a Met codon 5′ and in-frame with theantigen. Also, if desired, the carboxy-terminal or other region of theantigen can be removed using standard oligonucleotide mutagenesisprocedures.

The nucleotide (DNA) sequences can be expressed in hosts after thesequences have been operably linked to, i.e., positioned to ensure thefunctioning of, an expression control sequence. These expression vectorsare typically replicable in the host organisms either as episomes or asan integral part of the host chromosomal DNA. Commonly, expressionvectors can contain selection markers, e.g., tetracycline resistance orhygromycin resistance, to permit detection and/or selection of thosecells transformed with the desired DNA sequences (see, e.g., U.S. Pat.No. 4,704,362).

Host bacterial cells may be chosen that are mutated to be reduced in orfree of proteases, so that the proteins produced are not degraded. Forbacillus expression systems in which the proteins are secreted into theculture medium, strains are available that are deficient in secretedproteases.

Polynucleotides encoding a variant polypeptide may include sequencesthat facilitate transcription (expression sequences) and translation ofthe coding sequences such that the encoded polypeptide product isproduced. Construction of such polynucleotides is well known in the art.For example, such polynucleotides can include a promoter, atranscription termination site (polyadenylation site in eukaryoticexpression hosts), a ribosome binding site, and, optionally, an enhancerfor use in eukaryotic expression hosts, and, optionally, sequencesnecessary for replication of a vector.

Fermentation and Purification Procedures

This invention relates to improved methods of preparing B. anthracis PAfor use in vaccines. Procedures are exemplified herein for purifyingmodified PA from is a protease-deficient nonsporogenic avirulent strainof B. anthracis. However, it is expected that these procedures will beuseful for growing and purifying PA, including natural or recombinantPA, as well as various modified or truncated forms of PA, from othermicroorganisms, particularly Bacillus. Bacillus strains and/orexpression systems which are expected to be suitable include, forexample, the B. anthracis strain described in U.S. Pat. No. 5,840,312(Nov. 24, 1998) and the B. subtilis strain and PA expression systemdescribed in U.S. Pat. No. 6,267,966 (Jul. 31, 2001).

In the method of the invention, the culture is preferably maintained atabout pH 7 to about pH 8, most preferably about pH 7.5, substantiallythroughout the fermentation process. It has also been found to beadvantageous to add EDTA before separating the culture supernatant fromthe cells, preferably at or near the end of fermentation, since if it isadded during the fermentation stage, it may interfere somewhat with thegrowth of the cells.

The purification procedure of the invention is preferably essentially athree-step procedure, including (1) hydrophobic interactionchromatography, (2) ion exchange chromatography and (3) gel filtration.While ion exchange chromatography may precede hydrophobic interactionchromatography in the purification process, and still permit obtaining agood yield of PA, it is a less efficient process. Therefore, in view ofthis, it is preferred that hydrophobic interaction chromatographyprecede ion exchange chromatography in the purification process.Alternatively, this three step procedure need not be used and analternative purification scheme may be used.

In addition, the resins used in the exemplified the purificationprocedure can be substituted. For example, in the hydrophobicinteraction chromatography step, phenyl sepharose (Pharmacia) is used asthe resin in the example, but any other hydrophobic resin can be used.Likewise, in the ion exchange chromatography step, Q sepharose(Pharmacia) is used as the resin in the example, but any other anionexchanger can be used. Likewise, for the gel filtration step, Superdex(Pharmacia) is the residue used in the example, but it can be replacedby other gel filtration resins. Furthermore, with respect to thefermentation conditions, similar compounds can replace the tryptone andthe yeast extract that are obtained from Difco.

The expression and the stability of two recombinant PA variants,PA-SNKE-ΔFF-E308D and PA-N657A, were studied. However, the methods ofthe invention are also expected to be useful for producing andrecovering native PA; PA wherein the receptor-binding domain has beenaltered; PA which cannot be cleaved at the chymotrypsin cleavage site;PA which cannot be cleaved at the furin cleavage site; other PA whichcannot be cleaved at either the chymotrypsin or the furin cleavage sitein addition to the one exemplified herein (see, e.g., those described in(22)); PA fragments (e.g., a PA fragment having aa 175-764 (36)); PAmutants having a strong dominant-negative effect (e.g., PA doublemutants K397D and D425K) (37), and PA mutants with substitutions indomain 2 (37)).

In addition, the methods of the invention are also expected to be usefulfor producing and recovering PA in which the chymotrypsin site, FF, isreplaced by a furin site. This may be a suicide protein, getting easilycleaved by furin after binding to receptor. Cleavage at that siteinactivates PA.

The methods of the invention are also expected to be useful forproducing and recovering PA with a protease cleavage site (thrombin,Factor IV, etc.) at approximately residue 605. PA made in large amountsin the expression system could be cleaved to produce a soluble domain 4,which would compete with PA for receptor, and could be a therapeuticagent.

The methods of the invention are also expected to be useful forproducing and recovering PA with matrix metalloprotease or plasminogenactivator sites replacing the furin site (38, 39).

The methods of the invention are also expected to be useful forproducing and recovering other proteins, such as LF. See, e.g., (21),wherein expression system is the same, except the structural gene for PAis replaced by the LF gene. This can be generalized to include LFmutants altered in the catalytic site residues: HEFGH, 686-690. Thesystem may also have utility with EF.

The following examples are exemplary of the present processes andincorporate suitable process parameters for use herein. These parametersmay be varied, however, and the following should not be deemed limiting.

Example 1

In this example, the expression and the stability of two recombinant PAvariants, PA-SNKE-ΔFF-E308D and PA-N657A, were studied. These proteinswere expressed in the non-sporogenic avirulent strain BH445. Initialresults indicated that PA-SNKE-ΔFF-E308D, which lacks twoproteolysis-sensitive sites, is more stable than PA-N657A. Processdevelopment was conducted to establish an efficient production andpurification process for PA-SNKE-ΔFF-E308D. Various parameters such aspH, media composition, growth strategy, and protease inhibitorscomposition were analyzed. The production process chosen was based onbatch growth of B. anthracis using tryptone and yeast extract as theonly sources of carbon, pH control at 7.5, and antifoam 289. Optimalharvest time was found to be 14-18 hours after inoculation, and EDTA (5mM) was added upon harvesting for proteolysis control. In one of theprocesses described herein, recovery of the PA was performed by expandedbed adsorption (EBA) on a hydrophobic interaction resin, eliminating theneed for centrifugation, microfiltration, and diafiltration. The EBAstep was followed by ion exchange and gel filtration. PA yields beforeand after purification were 130 mg/L and 90 mg/L, respectively.

Materials and Methods Strains and Plasmids

The non-sporogenic, protease deficient, avirulent strain B. anthracisBH445 (pXO1⁻, pXO2⁻, cm^(r)) was used (17). The Bacillus-E. coli shuttlevector pYS5 (amp^(r), kan^(r))(26) was used to clone two recombinantforms of the protective antigen: N657A and SNKE-ΔFF-E308D (28). In theN657A mutant, the receptor-binding domain of PA was altered bysubstitution of Asp with Ala at position 657 (domain 4). In theSNKE-ΔFF-E308D mutant two regions were altered, the furin site (RKKR¹⁶⁷to SNKE¹⁶⁷) and the chymotrypsin site (two Phe at positions 313-314 weredeleted and Glu acid at position 308 was substituted with Asp). Both PAconstructs contain the DNA sequence encoding the signal peptide of PA.

Culture and Expression Conditions

Modified FA medium (21) containing (per liter) 35 g tryptone (DifcoLaboratories, Detroit, Mich.), 5 g yeast extract (Difco Laboratories),and 100 mL of 10× salts was used in all experiments. The 10× saltsolution (per liter) consisted of 60 g Na 2 HPO₄.7H₂O, 10 g KH₂PO₄, 55 gNaCl, 0.4 g L-tryptophan, 0.4 g L-methionine, 0.05 g thiamine, and 0.25g uracil. It was filter-sterilized and added to the fermentor aftercooling. The pH of the medium was adjusted to 7.5; 100 μg/mL kanamycinand 20 μg/mL chloramphenicol were added. Fermentation experiments wereperformed by inoculating a 12-14 hour-old starter culture grown from afrozen stock. The medium in the fermentor was supplemented with 0.2 mL/Lof antifoam 289 (Sigma, St. Louis, Mo.). Three- to ten-literfermentations were done using B. Braun Biostat MD DCU (Melsungen,Germany), controlling dissolved oxygen (DO) at 30% saturation,temperature at 37° C., and pH at 7.5 with HCl and NH₄OH. At harvesttime, 5 mM EDTA and 10 μg/mL PMSF (phenylmethyl sulfonyl fluoride) (inone of the experiments described herein) were added to the culture.Shake flask experiments (100 mL) utilizing modified FA medium weresupplemented with glucose, lactose, glycerol, and casitone at aconcentration of 10 g/L.

Analytical Methods

Optical density (OD) was measured at 600 nm. Protease analysis was doneon supernatant samples collected during growth and stored frozen at −20°C. EDTA was added to supernatant samples used for SDS-PAGE and radialimmunodiffusion to a final concentration of 10 mM.

Extracellular protease activity was detected using the EnzChek greenfluorescence assay kit (Molecular Probes Eugene, Oreg.). Fluorescencewas measured with a LS508 luminescence spectrophotometer (Perkin-ElmerBoston, Mass.). This assay was conducted at pH of 7.5 or 6.0 dependingon the experiment. Proteolytic activity is reported as fluorescencechange per unit sample.

Protein was determined using BCA assay (Pierce Rockford, Ill.). PAexpression was quantified by SDS-PAGE (Invitrogen/Novex, Carlsbad,Calif.) gel analysis and by the Mancini immunodiffusion assay (19) usingagarose plates containing polyclonal PA antibody. Pure PA was used asthe standard, both polyclonal PA antibodies and pure PA were supplied byS. Leppla

Purification

a. Packed Bed Hydrophobic Interaction Chromatography

The cell suspension containing 5 mM EDTA was centrifuged and thesupernatant passed through a 0.2 μm hollow fiber filter (AGT, Needham,Mass.). The filtered broth was then concentrated 20× using a 10Kmembrane in a Pellicon-2 (Millipore, Bedford, Mass.). 200 g (NH₄)₂SO₄per liter (1.5 M) were added to the concentrated supernatant. The smallamount of precipitate produced after addition of (NH₄)₂SO₄ waseliminated with centrifugation and filtration. Phenyl Sepharose FastFlow (Amersham Pharmacia Biotech) was equilibrated with buffercontaining 1.5 M (NH₄)₂SO₄/10 mM HEPES/5 mM EDTA pH=7.0 (equilibrationbuffer) at a flow rate of 15 cm/h. After sample loading, the column waswashed with 10 column volumes (CV) of equilibration buffer and PA waseluted with a 30 CV linear gradient from 1.5 M to 0 M (NH₄)₂SO₄ in 10 mMHEPES/5 mM EDTA; pH=7.0. Fractions were analyzed by SDS-PAGE and thePA-containing samples were pooled for further purification.

b. Expanded Bed Hydrophobic Interaction Chromatography

The cell suspension containing 5 mM EDTA was diluted 1:1 with buffercontaining 3.0 M (NH₄)₂SO₄/20 mM HEPES/5 mM EDTA and 0.005% PluronicF-68 (Life Technologies, Inc. Gaithersburg, Md.). STREAMLINE™ Phenyl,(Amersham Pharmacia Biotech) was expanded in a streamline column inequilibration buffer. The diluted cell suspension was loaded upward at300 cm/h. The column was washed in expanded mode (2) with 10 CV ofequilibration buffer containing 0.005% pluronic F-68. Elution wasperformed in packed bed mode with 8 CV of elution buffer at 100 cm/h.The eluent was analyzed by SDS-PAGE and radial immunodifussion.

c. Anion Exchange Chromatography

Fractions from HIC were dialyzed against 20 mM Tris pH=8.9 and loaded ona Q Sepharose Fast Flow (Amersham Pharmacia Biotech) column equilibratedwith 20 mM Tris pH=8.9 at 15 cm/h. The protein was eluted using a 20 CVlinear gradient from 0 to 0.5 M NaCl in the same buffer. PA containingfractions were concentrated and dialyzed against PBS.

d. Gel Filtration

The pooled PA was further purified using a Superdex 75 column (AmershamPharmacia Biotech) in PBS/5 mM EDTA pH=7.4 at 12 cm/h.

Results and Discussion

a. Expression of Two Recombinant PA: PA-N657A and PA-SNKE-AFF-E308D

The expression of two recombinant versions of PA and the extracellularproteolytic activity of the culture were analyzed (FIG. 1). Productionof PA-SNKE-AFF-E308D, the protein lacking the furin and chymotrypsincleavage sites, was nearly 60% higher than that of PA-N657A, the proteincontaining a mutation in the receptor-binding domain (FIG. 1 a). Theextracellular proteolytic activity (fluorescence/OD) of both cultureswas similar. SDS-PAGE analysis of partially purified PA recovered fromthese cultures shows higher concentration of smaller fragments in thesample from PA-N657A compared to the sample from PA-SNKE-AFF-E308D (FIG.1 b). Western blot analysis with polyclonal PA antibody confirmed thatthe smaller fragments were reactive against PA (data not shown). Asindicated in FIG. 1 a, the proteolytic activity was similar in bothstrains. Therefore, it was apparent that PA-SNKE-ΔFF-E308D is a bettercandidate, due to its stability, and it was selected for furtherstudies.

b. pH Effect

Based on previous information (5, 21), initial production studies withPA-SNKE-ΔFF-E308D were done by controlling pH with NH₄OH only, whichresulted in pH 8.7 at the end of the fermentation. When pH wascontrolled at 7.4 during the entire fermentation, the PA production was30 mg per g cell and the proteolytic activity per OD unit was 8,compared to values of 20 mg PA per g cells and proteolytic activity perOD of 30 when the pH control was done only by NH₄OH. When the processwas performed at a lower pH, both PA production and protease activitywere lower. At pH 6.1 production declined nearly six times and proteaseactivity two times compared to what was found at pH 7.4. Possibly,intracellular expression is lower or secretion is inhibited at low pH.From the above information it is obvious that pH significantly affectsthe proteolytic activity and the PA expression. Controlling pHthroughout the fermentation process resulted in a 30% increase in PAyield, compared to previously reported strategies.

c. Effect of Various Carbon Sources and Protease Inhibitors

Attempts to increase PA expression by supplementing the basic growthmedium with different carbon sources is summarized in Table 1.

TABLE 1 Effect of various carbon sources on PA production. PA productionMedium mg PA/g cell mg PA/L culture Basic medium 31.3 129.5 Glycerol +basic medium 23.7 117.3 Glucose + basic medium 25.3 113.3 Lactose +basic medium 33.9 116.0 Casitone + basic medium 28.3 135.1

Neither the volumetric production nor the production per gram cellscould be enhanced with the addition of various carbon sources. Theeffect of PMSF and EDTA on extracellular proteolysis was also examined.As shown in FIG. 2, addition of EDTA (15 mM) significantly reducedproteolytic activity whereas the proteolytic activity of thePMSF-containing fraction (1 g/mL) was similar to that of the control.Based on this information, EDTA was added at the end of thefermentation, before the protein was processed.

d. Growth and Production Conditions

Based on the parameters determined previously, a production process forthe recombinant PA-SNKE-ΔFF-E308D from B. anthracis BH445 wasestablished. The process is based on growth in a batch fermentationcontrolled at pH 7.5 with NH₄OH/HCl and at 30% dissolved oxygensaturation for a period of 18 hours. A typical fermentation is seen inFIG. 3.

In general, the final OD₆₀₀ values fluctuated between 16 to 20. Duringthe first five hours, growth was exponential and the pH was controlledby base addition. Later in the fermentation the pH was controlled byacid addition. Accumulation of PA occurred mostly during the stationaryphase and reached a final concentration of 160 mg per liter. The resultsshown in FIG. 4 indicate that PA degraded if the fermentation wasextended for more than 18 hours, therefore, a harvest time between 14and 18 hours was selected.

Attempts to increase the PA production by implementing a fed-batchgrowth strategy were conducted. The addition of 10× tryptone/yeastextract/salts or 50% glucose/10× salts resulted in a 50% increase incell density but not an increase in protein production (FIG. 5). Theobservations that PA production was not improved by the implementationof a fed batch growth strategy or by the addition of various carbonsources such as casein, glucose, glycerol or lactose is an indicationthat perhaps a specific nutritional factor is missing. It is alsoimportant to mention that the specific proteolytic activity was almostfive times lower when glucose was added to the tryptone/yeast extractmedia (FIG. 6). This was expected since glucose is known to be arepressor of proteases in Bacillus (10, 25).

e. Purification

The purification protocol developed for PA (Materials and Methods)consisted of hydrophobic interaction chromatography (Phenyl Sepharose)followed by anion exchange (Q Sepharose) and gel filtration (Superdex75). Replacing the initial capturing step with expanded bedchromatography (2) can simplify and shorten the recovery process sinceit eliminates the clarification steps. Therefore, the use of expandedbed adsorption (EBA) was investigated by substituting the traditionalpacked-bed resin (Phenyl Sepharose) with the expanded bed hydrophobicresin STREAMLINE™ Phenyl. The static binding capacity for STREAMLINE™Phenyl was approximately 15 mg protein/mL of resin which is comparableto the capacity of Phenyl Sepharose. Optimal binding of PA toSTREAMLINE™ Phenyl occurred at 1.5 M (NH₄)₂SO₄.

Preliminary experiments performed with cell-containing broth in expandedmode resulted in the formation of aggregates and eventual collapse ofthe bed. It was possible to stabilize the expanded column only after theaddition of a detergent which probably altered some of the hydrophobicinteractions but did not prevent PA from binding. Pluronic F-68 waschosen due its non-toxicity in humans. The static binding capacities ofSTREAMLINE™ Phenyl were 15, 11, and 5 mg protein/mL resin with 0%,0.005%, and 0.01% pluronic F-68, respectively. Successful operation ofthe HIC EBA column occurred when using a load concentration of 15 g wetcells/L, 0.8 mL resin/g wet cells, and 0.005% pluronic F-68 in the loadas well as the wash buffer. Under these conditions some signs ofaggregation appeared at the end of the loading phase but cell debris waseliminated in the washing phase. A 70% recovery was obtained.

PA purity after hydrophobic interaction chromatography was higher than80%. Further purification was achieved by adding gel filtration step(FIG. 6, Lane b). However, this material was not stable when stored at4° C. for three months (FIG. 6, Lane c). In contrast, pure and stable PAwas obtained after hydrophobic interaction chromatography on expandedbed, followed by anion exchange and gel filtration (FIG. 6, Lane d).Similar results to the expanded bed process were obtained when packedbed hydrophobic interaction chromatography was followed by ion exchangeand gel filtration (FIG. 6, Lane a).

Replacing the packed-bed capturing step with expanded bed adsorptionproved to be more efficient since it eliminated the centrifugation andfiltration steps, however, twenty times more (NH₄)₂SO₄ and three timesmore resin were required to process the same amount of culture (Table2).

TABLE 2 Comparison of packed bed and expanded bed absorption ascapturing processes for PA Packed Bed Expanded Bed Adsorption 1. Totalprocessing time 15.5 h 1. Total processing time: 8 h a) downstreamprocessing: 6 h a) downstream processing: 1 h (4 unit operations) (1unit operation) b) loading: 2 h b) loading: 4 h c) column wash: 3.5 h c)column wash: 1.5 h d) elution: 4 h d) elution: 1.5 h 2. 400 g (NH₄)₂SO₄needed 2. 8000 g (NH₄)₂SO₄ needed 3. 100 mL resin needed 3. 300 mL resinneeded 4. Load/wash steps require little attention 4. Load/wash stepscannot be left unattended 5. 82% recovery 5. 70% recovery

Initial work with hydrophobic interaction chromatography using expandedbed ad sorption to capture PA resulted in bed collapse. This was avoidedafter the addition of a surfactant (pluronic F-68). These resultssuggest that the characteristics of the cell membrane were most likelythe cause of cell aggregation. Since no polyglutamic acid capsule ispresent in the recombinant strain, the two hydrophobic membrane proteinsforming the S-layer (4, 6) may be responsible for associating withneighboring cell membranes and the resin. After evaluating the possibleinteractions affecting the system, it was found that successfuloperation of the expanded bed was possible by carefully adjusting thecell concentration of the load, increasing the adsorbent-to-cell ratio,and choosing the appropriate detergent type and concentration. Theexpanded bed approach was more efficient in spite of the slightly loweryield (70% vs. 82%) and the higher amount of (NH₄)₂SO₄ and resin neededsince it eliminated the need for centrifugation and filtration. Toobtain stable and highly purified protein, anion exchange and gelfiltration steps were added.

Conclusions

Once the gene encoding PA (pagA) was cloned (31) and sequenced (32),several researchers have reported on the expression of PA in hosts likeB. subtilis (1, 13, 20, 26), E. coli (8, 24, 31), Salmonella typhimurium(3), viruses (11), and avirulant B. anthracis (5, 15). From thesereports, the highest PA yield achieved has been in the order of 50 mg/Lin B. anthracis (15). In this work, a scalable fermentation andpurification process suitable for vaccine development which producedalmost three times more product than what have been reported earlier, ispresented. This was accomplished by using a biologically inactiveprotease-resistant PA variant in a protease-deficient nonsporogenicavirulent strain of B. anthracis.

Example 2 Composition of the Vaccines

Four combinations of the recombinant (modified) protective antigen(“rPA”) were made: (1) rPA in PBS (“phosphate buffered saline”), (2) rPAin formalin, (3) rPA in aluminum hydroxide and (4) rPA in formalin andaluminum hydroxide. Another formulation of succinylated rPA was preparedand tested (data not shown).

Example 3 Immunogenicity in Mice

The four formulations described above were immunogenic in mice, andinduced antibody levels comparable to those induced by the currentlylicensed anthrax vaccine. The induced antibodies had anthrax toxinneutralizing activity. It is planned to evaluate these formulations inhumans, and to chose the best one for use as a vaccine.

The data from the mice experiments are set forth in the tables 3 to 5below:

TABLE 3 Number of Mice and Immunogen Group Number Number of MiceImmunogen 1056 11 PA (2.5 μg)-Untreated 1057 11 PA (12.5 μg)-Untreated1058 11 PA (2.5 μg) + Alum 1059 10 PA_(SUCC) 10:1.25 (2.5 μg) 1060 10PA_(SUCC) 10:1.25 (12.5 μg) 1061 10 PA_(SUCC) 10:3 (2.5 μg) 1062 10PA_(SUCC) 10:3 (12.5 μg) 1063 10 PA-Formalin 0.3 (2.5 μg) 1064 10PA-Formalin 0.3 (12.5 μg) 1065 10 PA-Formalin 3.0 (2.5 μg) 1066 10PA-Formalin 3.0 (12.5 μg) 1067 10 PA-Formalin 7.12 (2.5 μg) 1068 10PA-Formalin 7.12 (12.5 μg) 1069 11 Anthrax Vaccine 0.1 ml 1070 10Control

TABLE 4 Antibody Levels and Neutralization Titers Mice μg/ml Neutral,Titer 1056A 130.64 4000 1056B 11.24 200 1056K 21.3 1000 1057A 146.653000 1057I 490.14 7000 1058A 725.31 8000 E 710.46 7000 J 513.46 40001059A 53.89 1500 1060A 125.92 850 1061A 97.1 1500 C 21.2 200 E 54.22 7001062A 24.9 1500 J 14.35 2000 1063A 68.31 1500 C 179.16 2000 H 564.942000 1064A 581.34 10,000 1064D 204.56 8000 E 742.21 11,000 F 418.95 7000G 814.91 10,000 1065A 77.73 1250 E 214.37 5000 1066C 65.47 4000 D 513.3210,000 E 248.91 4000 F 260.36 8000 J 1041.65 10,000 1067A 261.54 3000 G415 5000 1068A 512.99 10,000 I 414.82 5000 1069A 339.18 3000 1069J879.65 3000 1070E <.05 205-6 weeks old female general purpose mice were injected subcutaneouslywith 0.1 mL of the immunogens depicted in Table 3, 2 or 3 times 2 weeksapart. The mice were exsanguinated one week after the last injection andtheir sera assayed for IgG anti PA and anthrax toxin neutralization.Antibodies measured by Elisa were related to a standard containing 1.8mg/ml of anti-PA monoclonal antibody.

TABLE 5 IgG anti PA levels induced in mice by various rPA formulationsdose × number PA lot formulation of injections μg/ml 0 PA 2.5μ × 2 1.3 0PA 2.5μ × 3 109.1 2 PA 2.5μ × 3 24.9 2 PA 12.5μ × 3  226 0 PA/Al (OH)₃2.5μ × 2 86.1 0 PA/Al (OH)₃ 2.5μ × 3 312. 2 PA/Al (OH)₃ 2.5μ × 3 435. 2PA formalin 0.3 2.5μ × 3 182 2 PA formalin 0.3 12.5μ × 3  350. 0 PAformalin 3.0 2.5μ × 2 2.79 0 PA formalin 3.0 2.5μ × 3 136.4 0 PAformalin 3.0 5.0μ × 2 1.98 2 PA formalin 3.0 2.5μ × 3 220 2 PA formalin3.0 12.5μ × 3  270 0 PA formalin 7.12 2.5μ × 3 266 0 PA formalin 7.1212.5μ × 3  229 Anthrax Vaccine 1/10 human dose × 2 43.15 1/10 human dose× 3 297 PBS control ×2 <.05 ×3 <.055-6 weeks old female mice, 10 per group, were injected subcutaneouslywith the listed formulations, 2 or 3 times, two weeks apart andexsanguinated one week after the last injection. Antibodies weremeasured by Elisa, calculated relative to a standard containing 1.8mg/ml of anti-PA monoclonal antibody, and expressed as geometric meansof the groups.

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The disclosures of all the references cited hereinabove are incorporatedby reference herein.

Modifications of the above described modes for carrying out theinvention that are obvious to those of skill in the fields ofimmunology, protein chemistry, microbiology, medicine, and relatedfields are intended to be within the scope of the following claims.

1. An isolated modified Bacillus anthracis (B. anthracis) protectiveantigen, wherein the antigen comprises a B. anthracis protective antigenmodified such that the amino acid sequence RKKR¹⁶⁷ (SEQ ID NO: 14) hasbeen changed to SNKE¹⁶⁷ (SEQ ID NO: 15), the two phenylalanines atpositions 313-314 are deleted, and the glutamic acid at position 308 issubstituted with aspartic acid.
 2. The isolated protective antigen ofclaim 1, wherein the modified B. anthracis protective antigen cannot becleaved at the chymotrypsin cleavage site.
 3. The isolated protectiveantigen of claim 1, wherein the modified B. anthracis protective antigencannot be cleaved at the furin cleavage site.
 4. The isolated protectiveantigen of claim 1, wherein the protective antigen comprises the aminoacid sequence shown in SEQ ID NO:
 4. 5. A transformed host cellcomprising the protective antigen of claim
 1. 6. A pharmaceuticalcomposition comprising the protective antigen of claim 1 and aphysiologically acceptable carrier.
 7. The pharmaceutical composition ofclaim 6, wherein the physiologically acceptable carrier comprises salineor phosphate-buffered saline.
 8. A vaccine composition comprising atherapeutically effective amount of the protective antigen of claim 1and a pharmaceutically acceptable carrier.
 9. The vaccine composition ofclaim 8, wherein the physiologically acceptable carrier comprises salineor phosphate-buffered saline.
 10. The vaccine composition of claim 8,further comprising an adjuvant.
 11. The vaccine composition of claim 10,wherein the adjuvant comprises aluminum hydroxide.
 12. The vaccinecomposition of claim 8, further comprising formalin.
 13. The vaccinecomposition of claim 10, further comprising formalin.
 14. A vaccinecomposition comprising a therapeutically effective amount of theprotective antigen of claim 1 and a pharmaceutically acceptable carrierand aluminum hydroxide.
 15. A method for inducing serum antibodies whichhave neutralizing activity for B. anthracis toxin comprisingadministering to a mammal the pharmaceutical composition of claim 6comprising an amount of B. anthracis protective antigen sufficient toelicit production of said antibodies.
 16. The method of claim 15 whereinthe antibodies protect the human against infection by B. anthracis. 17.The method of claim 15 wherein the mammal is a human.
 18. A method forvaccinating a human against B. anthracis infection, comprisingadministering to the human an immunizing amount of the vaccinecomposition of claim
 8. 19. A method for vaccinating a human against B.anthracis infection, comprising administering to the human an immunizingamount of the vaccine composition of claim
 11. 20. A method forvaccinating a human against B. anthracis infection, comprisingadministering to the human an immunizing amount of the vaccinecomposition of claim 14.